DNA extraction from tissue protocol

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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="DNA extraction buffer">DNA extraction buffer <i><br><tab><div style="margin-right: 600px;">(98µl ReagentB + 2µl ProteinaseK, mix fresh)</div></i></a></li><li>24:1 Chloroform:Isoamyl alcohol</li><li> <a name="PCI mix">PCI mix <i><br><tab><div style="margin-right: 600px;">(One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion)</div></i></a></li><li>100% EtOH</li><li>3M sodium acetate pH 5.0</li><li>70% EtOH</li><li>MilliQ water</li><li>small piece of tissue or embryo</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Proteinase K digestion</font></b><br><ol type="a"><p><li>Measure out <b><font color=#357EC7>100 µl</font></b> of <a href="#DNA extraction buffer" ><font color=#357EC7>DNA extraction buffer</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br><font color = "#800517"><i>100 µl is enough for a small pea size chunk of tissue or one embryo.</i></font><br></li></p><p><li>Add <font color=#357EC7>small piece of tissue or embryo</font>.<br></li></p><p><li>Incubate at <b><font color=#357EC7>50°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p></ol></li></p><p><li><b><font size=3>Phenol/chloroform/isoamyl (PCI)</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>1</font></b> volume <a href="#PCI mix" ><font color=#357EC7>PCI mix</font></a>.<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).<br>Discard bottom layer.<br><font color = "red"><i>Repeat this step as needed.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>1</font></b> volume <font color=#357EC7>24:1 Chloroform:Isoamyl alcohol</font> to sterile 1.5-ml microcentrifuge tube (2).<br></li></p><p><li>Vortex the mixture for <b><font color=#357EC7>10 secs</font></b> .<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).<br>Discard bottom layer.<br></li></p></ol></li></p><p><li><b><font size=3>Ethanol precipitation</font></b><br><ol type="a"><p><li>Add <b><font color=#357EC7>2</font></b> volumes <font color=#357EC7>100% EtOH</font> to sterile 1.5-ml microcentrifuge tube (3).<br></li></p><p><li>Add <b><font color=#357EC7>0.1</font></b> volume <font color=#357EC7>3M sodium acetate pH 5.0</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <font color=#357EC7>70% EtOH</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Dry the pellet in air. <br></li></p><p><li>Add <font color=#357EC7>MilliQ water</font> to pellet.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 12 hrs, 22 mins</font></b></p> </html>